This invention relates to transgenic fish containing a modified fish insulin gene which has been altered to produce humanized insulin. This invention further relates to improved methods for the xenotransplantation of transgenic islets in the treatment of diabetes. In a further aspect, the present invention relates to improved methods for mass isolation of fish islets.
Diabetes mellitus is a disease resulting in significant morbidity and mortality. The total annual direct and indirect costs of diabetes in the Unites States exceeds $90 billion dollars. Insulin-dependent diabetes mellitus (IDDM), because it occurs in a younger population than non-IDDM, accounts for a disproportionate percentage of these costs. Although the acute manifestations of IDDM can be controlled with daily insulin injections, most patients eventually develop sequelae such as blindness, nephropathy, neuropathy, microangiopathy, and cardiovascular disease. Substantial evidence suggests that meticulous control of glycemia will prevent or minimize these sequelae.
A more physiological method of treating diabetes would be pancreas or islet transplantation. Whole or segmental pancreas transplantation has been performed successfully in man and some preliminary evidence suggests that this technique will prevent the sequelae of diabetes in man. However, pancreas transplantation is not trivial surgery; it poses problems with drainage of exocrine secretions and requires a lifetime of immunosuppressive therapy. On the other hand, islet transplantation has certain theoretical advantages, particularly related to the ease of surgery, the absence of extraneous exocrine tissue, and the cryopreservability of isolated islets. More importantly, islets are more amenable to immunoalteration. Indeed, various methods have been developed to prolong allograft survival without continuous immunosuppression in rats and mice. The ability to transplant islets without continuous immunosuppression may eventually prove absolutely necessary in man because many immunosuppressive drugs are somewhat toxic to islets.
Recent improvements in the methods of mass islet isolation and several recent clinical reports suggest that islet transplantation is on the verge of becoming a feasible treatment for IDDM. However, several obstacles exist. First, islets comprise only 2% of the human pancreas; yields from human xe2x80x9cislet isolationxe2x80x9d procedures are extremely variable and several human donor pancreases are often required to generate sufficient islets for a single transplant. Thus, there are insufficient human donor pancreases available to treat the vast numbers of type I diabetic patients. Second, islet allograft rejection has proven difficult to manage using conventional methods and, unfortunately, the majority of islet allografts are quickly lost. Therefore, it seems likely that widespread implementation of islet transplantation would require the development of islet xenotransplantation methods.
In response to this eventuality, many biomedical corporations are spending millions of dollars developing and patenting xe2x80x9cbio-artificial pancreasxe2x80x9d technologies (i.e., microencapsulation or macroencapsulation of islet tissue). The underlying concept behind these approaches is that the islet tissue is protected from the immune system by a membrane with pore sizes small enough to prevent immunocytes and antibodies from damaging the graft yet large enough for insulin, oxygen, glucose, and nutrients to pass freely.
During the past few years, several clinical islet transplantation centers have devoted extensive effort to the development of experimental islet xenotransplantation models using large animals as donors. Most of these studies have centered on porcine, bovine, canine, or non-human primate islets. However, the pancreata in these species, like the human pancreas, are fibrous and do not readily yield large quantities of intact, viable islet tissue. Moreover, generation of islet preparations from large animal donors is expensive and islet yields are variable.
Brockmann bodies are anatomically discrete organs in certain teleost fish. Teleost fish insulin has been used in certain cases to maintain human diabetics (Wright, J R Jr., Experimental transplantation using principal islets of teleost fish Brockmann Bodies. Pancreatic Islet Cell Transplantation: 1892-1992xe2x80x94One Century of Transplantation for Diabetes, edited by C. Ricordi, R. G. Landes Co., Austin, 1992, p. 336-351). However, it is likely that the immunogenicity of teleost insulin may prevent clinical application for BB xenotransplantation. On the other hand, the production of transgenic fish whose BBs produce humanized insulin may circumvent this problem. Transgenic fish having BBs that physiologically secrete humanized insulin would eliminate the need for human pancreatic donors and procedures for the isolation of islets therefrom.
Until recently, BBs were harvested manually by microdissection while being visualized through a dissecting microscope inside a laminar flow hood (Wright J R Jr. Preparation of Fish Islets (Brockmann bodies) In: Lanza R P; W L Chick, eds. Pancreatic Islet Transplantation genes Vol. 1. Procurement of Pancreatic Islets. Austin: RG Landes co.; (1994:123-32)). While this is much easier and less expensive than the standard procedure of harvesting islets from rodents, it is a time consuming and tedious task. Although it is easy to harvest sufficient islets to perform xenografts in mice, this method is not well suited to harvest large volumes of islet tissue as would be required for clinical use or large animal studies. Furthermore, microdissection allows collection of less than 50% of the islet tissue per donor fish (i.e., those large BBs that are easily visible with the naked eye). Therefore, development of a more efficient method of harvesting BBs would be critical for the future application of fish islets as a donor source for clinical and experimental use.
To date, transgenic fish technology has been used to produce hardier fish that will grow rapidly and will tolerate adverse environments (Hackett P B: The molecular biology of transgenic fish. In: Biochemistry and molecular Biology of Fishes 2, Hochachka P and Mommesen T (eds.) Amsterdam: Elsevier, 1993; and Hackett P B: The molecular biology of transgenic fish. In: Biochemistry and molecular Biology of Fishes 2, Hochachka P and Mommesen T (eds.) Amsterdam: Elsevier, 1993). Most of these efforts have been directed at insertion of growth hormone transgenes. Another approach has been to insert antifreeze genes from species that tolerate very cold waters (i.e., such as winter flounder) into other species so that they will not only survive, but actually thrive in colder water. This approach permits aquaculture in more northerly regions and allows aquaculture stocks to grow year-round, rather than just during the summer growth season.
Accordingly, there exists a need for a method to supplement human islets with islets from other species without provoking an immune response. In addition, there exists a need in the art for improved methods of harvesting islets for use in xenotransplantation.
The objectives of the present invention are to provide a genetically stable production strain of transgenic tilapia for use in tissue transplantation. This line should have the following properties: (1) stable integration of humanized insulin gene in Teleost fish, (2) production of insulin in transgenic Teleosts under physiological glucose regulation, (3) absence of humanized tilapia DNA sequences other than the insulin locus, (4) homozygosity for the humanized insulin gene at the tilapia insulin locus, (5) genetic and developmental stability and uniformity, (6) good growth and survival characteristics, and (7) genetic identifiability for security against contamination and for protection of the proprietary interests of the developers.
These and other objects of the invention will become apparent upon review of the following description of the invention and appended claims.
This invention relates to a humanized tropical fish insulin gene capable of being physiologically expressed in a tropical fish islet cell. By virtue of the similarity between fish and human insulin, it has been possible to modify a fish insulin gene to encode a protein having the same alpha and beta chains as a human insulin while using fish-preferred codons. The humanized fish gene thus comprises a coding sequence of a humanized insulin gene under the control of regulatory sequences of the tropical fish insulin gene. In this manner, the protein processing machinery of the fish islet cell operates to process the initial translation product to yield a fully-formed humanized insulin polypeptide that is biologically active and substantially non-immunogenic in humans.
In another aspect, the present invention also relates to the preparation of transgenic tropical fish whose islet cells secrete humanized insulin. In a preferred embodiment, the tropical fish is a teleost fish such as tilapia.
In another embodiment, the present invention is directed to a unique animal model for islet xenotransplantation utilizing tilapia, a teleost fish, as islet donors (Wright, J R Jr., supra). The islet tissue in certain teleost fish, called principal islets or Brockmann bodies (BBs), is anatomically distinct from their pancreatic exocrine tissue and can be easily identified macroscopically. Thus, the islets can be readily isolated using straightforward techniques rather than expensive islet isolation procedures, such as is required when procuring islet tissue from mammalian pancreases. Tilapia islets transplanted into diabetic nude mice have been observed to produce long-term normoglycemia and a mammalian-like glucose tolerance curve (Wright, J R Jr., Polvi S. and Maclean H: Experimental transplantation using principal islets of teleost fish (Brockmann bodies): Long-term function of tilapia islet tissue in diabetic nude mice. Diabetes 41:1528-32.1992).